Biotransformation of a potent anabolic steroid, mibolerone, with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina, and biological activity evaluation of its metabolites.

Seven metabolites were obtained from the microbial transformation of anabolic-androgenic steroid mibolerone (1) with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina. Their structures were determined as 10ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (2), 6ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (3), 6ß,10ß,17ß-trihydroxy-7α,17α-dimethylestr-4-en-3-one (4), 11ß,17ß-dihydroxy-(20-hydroxymethyl)-7α,17α-dimethylestr-4-en-3-one (5), 1α,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (6), 1α,11ß,17ß-trihydroxy-7α,17α-dimethylestr-4-en-3-one (7), and 11ß,17ß-dihydroxy-7α,17α-dimethylestr-4-en-3-one (8), on the basis of spectroscopic studies. All metabolites, except 8, were identified as new compounds. This study indicates that C. blakesleeana, and C. echinulata are able to catalyze hydroxylation at allylic positions, while M. phaseolina can catalyze hydroxylation of CH2 and CH3 groups of substrate 1. Mibolerone (1) was found to be a moderate inhibitor of ß-glucuronidase enzyme (IC50 = 42.98 ± 1.24 µM) during random biological screening, while its metabolites 2-4, and 8 were found to be inactive. Mibolerone (1) was also found to be significantly active against Leishmania major promastigotes (IC50 = 29.64 ± 0.88 µM). Its transformed products 3 (IC50 = 79.09 ± 0.06 µM), and 8 (IC50 = 70.09 ± 0.05 µM) showed a weak leishmanicidal activity, while 2 and 4 were found to be inactive. In addition, substrate 1 (IC50 = 35.7 ± 4.46 µM), and its metabolite 8 (IC50 = 34.16 ± 5.3 µM) exhibited potent cytotoxicity against HeLa cancer cell line (human cervical carcinoma). Metabolite 2 (IC50 = 46.5 ± 5.4 µM) also showed a significant cytotoxicity, while 3 (IC50 = 107.8 ± 4.0 µM) and 4 (IC50 = 152.5 ± 2.15 µM) showed weak cytotoxicity against HeLa cancer cell line. Compound 1 (IC50 = 46.3 ± 11.7 µM), and its transformed products 2 (IC50 = 43.3 ± 7.7 µM), 3 (IC50 = 65.6 ± 2.5 µM), and 4 (IC50 = 89.4 ± 2.7 µM) were also found to be moderately toxic to 3T3 cell line (mouse fibroblast). Interestingly, metabolite 8 showed no cytotoxicity against 3T3 cell line. Compounds 1-4, and 8 were also evaluated for inhibition of tyrosinase, carbonic anhydrase, and α-glucosidase enzymes, and all were found to be inactive.

Identification of early response genes and pathway activated by androgens in the initial segment and caput regions of the regressed rat epididymis.

To identify the initial response to androgens and estrogens in the orchidectomized, regressed epididymis, we determined the gene expression changes triggered by the administration of either of two metabolites of testosterone, 5alpha-dihydrotestosterone (DHT) or 17beta-estradiol (E2), in the regressed rat epididymis. Adult rats were orchidectomized and 8 d later implanted with either empty implants (control), DHT-filled-, or E2-filled-polydioxanone implants. Rats were euthanized 12 h, 1 d, and 7 d later, and RNA was extracted and probed on Rat230-2.0 Affymetrix arrays. Probe sets that respond to DHT or E2 were identified at early time points; although the expression of some was repressed, the expression of many others was either transiently or chronically elevated. Nerve growth factor receptor (Ngfr) and S100 calcium binding protein G (S100g) were two E2 up-regulated genes detected at 12 h. Among the genes that showed a dramatic early response to DHT were endothelin 1 (Edn1), bone morphogenetic protein 4 (Bmp4), and IGF binding protein 3 (Igfbp3), which were suppressed, and IGF-I (Igf1), which was induced. Genes that were up- or down-regulated by DHT were classified based on biological function. Using PathwayStudio 4.0, we identified genes that were linked and directly influenced either the expression or regulation of one another. Epidermal growth factor and IGF-I play an important role in the pathway due to their function in regulation and expression of many other genes. These results provide novel insights into the impact of androgen action on the expression of genes that are important for epididymal function.

New steroidal aromatase inhibitors: suppression of estrogen-dependent breast cancer cell proliferation and induction of cell death.

BACKGROUND: Aromatase, the cytochrome P-450 enzyme (CYP19) responsible for estrogen biosynthesis, is an important target for the treatment of estrogen-dependent breast cancer. In fact, the use of synthetic aromatase inhibitors (AI), which induce suppression of estrogen synthesis, has shown to be an effective alternative to the classical tamoxifen for the treatment of postmenopausal patients with ER-positive breast cancer. New AIs obtained, in our laboratory, by modification of the A and D-rings of the natural substrate of aromatase, compounds 3a and 4a, showed previously to efficiently suppress aromatase activity in placental microsomes. In the present study we have investigated the effects of these compounds on cell proliferation, cell cycle progression and induction of cell death using the estrogen-dependent human breast cancer cell line stably transfected with the aromatase gene, MCF-7 aro cells. RESULTS: The new steroids inhibit hormone-dependent proliferation of MCF-7aro cells in a time and dose-dependent manner, causing cell cycle arrest in G0/G1 phase and inducing cell death with features of apoptosis and autophagic cell death. CONCLUSION: Our in vitro studies showed that the two steroidal AIs, 3a and 4a, are potent inhibitors of breast cancer cell proliferation. Moreover, it was also shown that the antiproliferative effects of these two steroids on MCF-7aro cells are mediated by disrupting cell cycle progression, through cell cycle arrest in G0/G1 phase and induction of cell death, being the dominant mechanism autophagic cell death. Our results are important for the elucidation of the cellular effects of steroidal AIs on breast cancer.

Synthesis and biological properties of 17 alpha-furylestradiol and dihydroequilin derivatives.

A series of 17alpha-furylestradiol and dihydroequilin derivatives was synthesized by reacting the appropriate 3-substituted estrone and equilin with 2- or 3-furyllithium. The oral estrogenic activity of the compounds was compared with that of mestranol. In the Allen-Doisy test, the 17alpha-(3-furyl) analogs were 4-19 times as potent orally as the standard in rats but they were less active in mice. Acetylation of the 17-alcohol or replacement of the 3-furyl by a 2-furyl group produced a decrease in activity. In the mouse uterotrophic assay in mice the compounds were less effective than mestranol and exhibited very shallow dose-response curves.

Radioautographic study of the effect of estradiol-17 , estrone, estriol, progesterone, testosterone and corticosterone on the in vitro uptake of 2,4,6,7- 3 H estradiol-17 by uterine eosinophils of the rat.

PIP: The in vitro uptake of 2,4,6,7-tritiated estradiol-17beta in uterine eosinophils of the rat was inhibited by the presence of nonradioactive estradiol-17beta, estrone, and estriol, but not by progesterone, testosterone, or corticosterone. This action is attributed to competition between tritiated estradiol and the various estrogenic compounds for the same binding site. Compounds without any estrogenic activity do not compete. The proposal is made that the eosinophil binding system and the 8S-5S binding system are involved in different mechanisms of estrogen action. The parallelism between the doses of estradiol and estriol needed to promote certain estrogenic early effects in the uterus, and the affinity of these steroids for the eosinophil uptake sites, suggests that uterine eosinophils might be responsible for some of these early effects, such as water imbibition, histamine releasing activity, and estrogen priming effect.^ieng

Further changes in differentiation state accompanying the conversion of Chinese hamster cells of fibroblastic form by dibutyryl adenosine cyclic 3':5'-monophosphate and hormones.

The morphological conversion in vitro of Chinese hamster ovary cells to a fibroblast form by a relatively large amount of dibutyryl adenosine cyclic 3':5'-monophosphate, or by a combination of small amounts of this compound and testosterone, is attended by appearance of the following additional properties: acquisition of strict contact inhibition of growth; reorientation of the random growth pattern into one in which cells grow parallel to their long dimension; disappearance of the randomly distributed, knob-like, pseudopodal structures around the cell periphery; induction of collagen synthesis; and decrease in the ability to be agglutinated and rounded up by plant agglutinins and specific cell antibodies. The changes in these characteristics are consistent with the conversion from a malignant to a normal fibroblastic state. This conversion is under genetic control, as demonstrated by the production of specific mutants with altered characteristics. The response to testosterone is specific since steroids like estradiol and hydrocortisone are inactive, and others have limited activity. Some prostaglandins are equal in activity to testosterone and 5alpha-dihydrotestosterone. This system appears useful in study of the regulation of phenotypic expression in mammalian cells.